Project Summary/Abstract The Wnt/?-catenin signaling pathway regulates cell growth and proliferation during organism development and in adulthood. Dysregulation of this pathway contributes to the progression of multiple diseases, including bone and vascular disorders, diabetes, and several types of cancer. Secreted Wnt proteins act via the transmembrane receptors of the Frizzled (Fzd) family and the co-receptor lipoprotein receptor-related protein 5 or 6 (LRP5/6) to recruit the cytoplasmic protein Dishevelled (Dvl) and ultimately stabilize the transcriptional co-activator ?-catenin to promote gene transcription. Although Wnt/?-catenin signaling has been well studied in model organisms and cell culture, molecular-level understanding of signal transduction in this therapeutically relevant pathway remains incomplete. Dvl is known to polymerize at the plasma membrane and to interact directly with specific membrane lipids, but the mechanisms by which these interactions contribute to ?-catenin stabilization are not completely understood. Here, we propose to investigate the initial steps of Wnt/?-catenin signaling by completing the following aims: Aim 1. Determine the individual contributions of Wnt, Fzd, and Dvl to LRP6 phosphorylation Dvl-promoted phosphorylation of the Wnt co-receptor LRP5/6 is a key step leading to ?-catenin-mediated transcription. Using purified proteins reconstituted into lipid bilayer nanodiscs, we will test the model that Dvl plays a crucial role in promoting LRP5/6 phosphorylation by concentrating receptors together with the scaffolding protein Axin on the plasma membrane. Aim 2. Examine the influence of membrane composition on Fzd and Dvl function The presence of specific lipids or cholesterol in cell membranes is known to affect the function of membrane proteins, and specific domains of Dvl have been reported to interact with membrane lipids. We will reconstitute signaling complexes in nanodiscs of defined lipid composition to test the hypothesis that local membrane environment alters multiple key interactions in Wnt/?-catenin signaling. Successful completion of these aims will provide molecular-level insights into the initiating steps of Wnt/?-catenin signaling. A full understanding of Wnt/?-catenin signaling is crucial for the development of safe, effective therapeutics targeting this pathway, therefore the knowledge provided by this work will aid in the development of novel targeted agents for treatment of multiple diseases.